KR20100090731A - Immunoglobulin preparations having increased stability - Google Patents

Abstract

The present invention relates to an increased protein formulation comprising a stabilizer selected from the group consisting of nonpolar and basic amino acids and having a pH of 4.0 to 5.2. The present invention also relates to pharmaceutical compositions and methods for stabilizing protein preparations.

Description

Immunoglobulin preparations having increased stability

The present invention relates to an increased protein formulation comprising a stabilizer selected from the group consisting of nonpolar and basic amino acids and having a pH of 4.2 to 5.4. The present invention also relates to pharmaceutical compositions and methods for stabilizing protein preparations.

Protein preparations, particularly immunoglobulin preparations for intravenous injection, have been used for some time. Proteins, particularly immunoglobulins, tend to form aggregates and / or dimers and fragment or denature. If such a solution is injected intravenously, aggregates can cause serious side effects, such as anaphylactic shock. Numerous measures have been attempted in the art to avoid such aggregation and fragmentation in such protein solutions and to improve their stability. For example, IgG for intravenous use in the clinic is often lyophilized to improve storage stability, but such formulations must be reconstituted with diluents prior to use. The reconstitution step is inconvenient, time-consuming and increases the likelihood of contamination of the product. Another method well known in the art for improving immunoglobulin stability and storage is the addition of protein-stabilizing excipients to IgG preparations. Known excipients include sugars, polyols, amino acids, amines, salts, polymers and surfactants. Taking such stabilization strategies in protein pharmaceuticals is common in the art. For example, US Pat. No. 4,499,073 (Tenold) improves stability through the selection of pH and ion concentration. JP 54020124 describes the addition of amino acids to intramuscular formulations in order to impart storage stability and safety. JP 57031623 and JP 57128635 describe the use of arginine and / or lysine with NaCl in 5-15% IgG preparations to achieve long term stability of intramuscular preparations. JP 56127321 describes the addition of sugar alcohols to IgG, which addition works better than the glucose previously used to inhibit aggregation. JP 4346934 describes the use of low conductivity (less than 1 mmho), pH 5.3 to 5.7, and optionally one or more stabilizers such as PEG, human serum albumin and mannitol. US Pat. No. 4,439,421 (Hooper) teaches the addition of hydrophilic polymers, polyols and other proteins to stabilize against ACA (anti-complementary activity) production. US Pat. No. 5,945,098 (Sarno) describes stabilization of isotonic solutions by addition of amino acids (0.1-0.3 M glycine), and non-ionic detergents (polysorbates) and PEG. US Patent No. 4,186, 192 (Lundblad) describes various additives, including amino acids, but does not specify the use of one particular amino acid. Such substrates include stabilization of IgG with maltose and further up to 0.1 M of glycine. No. 4,362, 661 (Ono) describes the use of neutral and basic amino acids to impart stability to 5% IgG products. All of the documents mentioned above describe an IgG preparation of still relatively high pH, which is acidic, but above 5.2.

In addition to preventing the formation of immunoglobulin aggregates, it has been recognized that dimer formation of IgG in particular may be detrimental to IgG preparations for intravenous use. Although IgG dimers are not known to cause anaphylactic shock, IgG preparations with high dimer content are much less tolerant upon intravenous injection, which can cause undesirable side effects including fever, nausea and sometimes lower blood pressure. Hypotension side effects were found in the rat model of Bleaker et al., Vox Sanguins 52,281-290, 1987, which also shows a clear correlation with dimer content. When lyophilized immediately after the preparation of the IgG preparation, dimer formation is not a major problem. However, if the formulation is to be stored in a liquid form that is not lyophilized, the dimer concentration increases with storage time.

U.S. Patent 5,871,736 (Bruegger et al.) Discloses immunoglobulin formulations, in particular liquid formulations of IgG for intravenous infusion, comprising at least one amphiphilic stabilizer for stabilization against dimer formation. Listed. Both lipophilic stabilizers include nicotinic acid and derivatives thereof, in particular nicotinamide, and amino acids with uncharged lipophilic side chains, mainly together with the foregoing, for example phenylalanine, methionine, leucine, isoleucine, proline and Valine is included. According to the experiments described in the prior art document, amino acids are always present with nicotinamide, the concentrations described for amino acids are 200 mmol / l for proline, 80 mmol / l for glycine and 120 mmol / l for isoleucine to be.

The pH range for the formulations described in US Pat. No. 5,871,736 is broadly given as 4-8, but what is actually described in the Examples teaches pH 5.3.

Although the above US patent describes IgG preparations in which dimer formation is inhibited to a certain level, it is still desirable to provide protein preparations, especially immunoglobulin preparations, which have improved stability at ambient temperatures.

Surprisingly, the inventors of the present application have found that a high degree of stabilization of liquid protein formulations can be achieved by adjusting the pH of the final formulation to 4.2 to 5.4 and adding basic or nonpolar amino acids as stabilizers.

Accordingly, the present invention provides protein formulations with improved stability, comprising one or more stabilizers selected from the group consisting of nonpolar and basic amino acids. Examples of nonpolar and basic amino acids useful for the purposes of the present invention are histidine, arginine, lysine and ornithine (basic amino acids), and isoleucine, valine, methionine, glycine and proline (non-polar amino acids). In particular, proline is useful. Stabilizers may be only amino acids of one of the nonpolar or basic amino acid groups, or a combination of two or more such amino acids. The amino acids are preferably not used with nicotinamide. Amino acid stabilizers can be natural amino acids, amino acid analogs, modified amino acids or amino acid equivalents. L-amino acids are preferred. If proline is used as a stabilizer, L-proline is preferred. Proline equivalents, for example proline analogs, can also be used.

Surprisingly, it has been found that adding only the amino acids themselves and adjusting the pH of the final formulation, without other stabilizers (eg nicotinamide), significantly increases the stability of these formulations, especially at ambient temperature. The increased stability is a temperature of about 2 ° C. to about 40 ° C., particularly preferably about 10 ° C., more preferably about 15 ° C., even more preferably about 20 ° C. to about 30 ° C., and most preferably about 25 ° C. This is evidenced by the good stability of the formulation at ambient temperatures in the range of < RTI ID = 0.0 > The increased stability of the formulations of the present invention is also apparent at high temperatures of about 30 ° C to about 40 ° C, including body temperatures of about 37 ° C. Preferably, increased stability is also alternatively or further defined as increased storage time, reduced fragmentation, reduced aggregate formation, reduced dimer formation and / or reduced bleaching. The improved storage time is preferably such that the formulation of the invention is at least about 30 days, preferably at least 60 days, more preferably at least 90 days, more preferably at least 120 days, even more preferably longer than this. Means stable while.

Reduction of aggregation preferably means that the formulation shows a lower percentage of aggregates than the conventional formulation (particularly for Ig). Preferably, the dimer content of the formulation is about 12% or less, preferably about 10% or less, more preferably about 8% or less. The reduction in coloration preferably means that the optical density of the formulations of the present invention is about 20% to 60% lower than conventional formulations.

In general, the protein preparations of the invention are liquid formulations useful for intravenous injection. Such formulations can be stored in liquid form and are stable and therefore do not require lyophilization or other measures and can be readily used.

Preferably, the protein preparation is an immunoglobulin preparation, in particular an antibody preparation wherein the antibody can be of any idiotype, but preferably is IgG, IgA or IgM. IgG preparations are particularly preferred. Immunoglobulins can be polyclonal or monoclonal, can be isolated from human or animal blood, or can be prepared by other means, such as recombinant DNA techniques or hybridoma techniques. In general, immunoglobulins are obtained from plasma by alcohol fractionation, which can be combined with other purification techniques such as chromatography, adsorption or precipitation. The immunoglobulins can be treated with trace enzymes (eg pepsin) to reduce anti-complementary activity, or they can be used as is.

The formulation can be obtained by methods known in the art, except that the pH of the final formulation is adjusted to a relatively high but acidic pH, ie, in the range of about pH 4.2 to 5.4. It has been found that this pH range is particularly useful for improving the storage characteristics of immunoglobulin formulations. The pH range is preferably 4.5 to about 5.2, a pH range of about 4.6 to 5.0 is particularly preferred, and pH 4.8 is particularly preferred.

In the course of developing the preparations according to the invention, it has also been found that increasing the final concentration of stabilizer can lead to surprising improvements in the storage characteristics and stability of the preparations. Thus, stabilizers are added at final concentrations of at least 0.2 M. Preferably, the final concentration is 0.2 M to 0.4 M, more preferably 0.2 M to 0.3 M and most preferably 0.25 M.

The present invention particularly relates to protein preparations with relatively high protein concentrations. The protein concentration of the final formulation of the invention is about 5-25% w / v, preferably about 6-15% w / v, more preferably about 8-12% w / v, most preferably about 10% w / v. Final protein concentration will depend on various factors, such as the route of administration, the type of condition to be treated, and the like. One skilled in the art will be able to determine the optimal protein concentration for the intended application. For example, for intravenous infusion, it is preferred that the final formulation of the invention has a protein concentration of about 15-20% w / v, preferably about 8-12% w / v. In the case of IgG for intravenous use, 10% w / v, ie 100 g IgG / l is particularly useful. For subcutaneous administration, higher doses may be chosen, for example about 15-20% w / v.

The present invention also provides a pharmaceutical composition comprising the protein formulation of the invention and a pharmaceutically acceptable additive. Such additives may be excipients, diluents such as water, and other materials such as non-buffering materials, for example sodium chloride, glycine, sucrose, maltose and sorbitol. Such pharmaceutical compositions can be administered by a variety of routes. For intravenous administration, a dose of about 0.2 g, preferably 0.5 g to about 2.0 g, of immunoglobulin per kg body weight per day may be used.

Another aspect of the present invention provides a protein preparation, in particular providing an aqueous protein solution and adding one or more stabilizers selected from the group consisting of basic and nonpolar amino acids to adjust the pH of the solution to about 4.2 to 5.4 It is a method of stabilizing immunoglobulin preparations. The pH is preferably adjusted to a value within the preferred range defined above, with pH 4.8 being particularly preferred. The method preferably comprises adjusting the protein concentration and stabilizer concentration and selecting the stabilizer (s) as described above (proline is particularly preferred).

In particular, the method provides an aqueous protein solution having a protein concentration of about 5-25% w / v, adjusts the pH of the solution to 4.2 to 5.4, and adds one or more stabilizers selected from the group described above to the solution. Thereby bringing the final stabilizer concentration to 0.2-0.4 M, to obtain a stable protein preparation. Numerous methods are known for separating immunoglobulins from human plasma or fractions thereof. Immunoglobulins can be purified, for example, by cold ethanol fractionation and / or octanoic acid fractionation and / or chromatography. Particularly preferred purification methods for the purposes of the present invention include ethanol fractionation, followed by octanoic acid fractionation, followed by low pH treatment, chromatography and nanofiltration. In preparing immunoglobulins for intravenous use as in the present invention, particular care is taken to reduce or eliminate immune complexes with anti-complementary activity and proteases such as kallikrein or plasminogen. It is desirable to. The proteins used in the protein preparations of the invention can be brought to the desired concentration of about 5-25% w / v by known methods, for example by ultrafiltration. The pH of the liquid protein preparation is adjusted to a pH of 4.2 to 5.4 and the stabilizer is added to the solution at a final concentration of about 0.2 M or more. Preferably, proline is used as a stabilizer, which is preferably added at a concentration of about 0.2 M to 0.4 M, preferably about 0.25 M.

The invention will now be described by the following examples and figures.

FIG. 1 shows aggregate content measured by HPLC for 8% IgG solution containing 0.25 M proline or 0.25 M glycine.
2 shows the dimer content measured by HPLC for 8% IgG solution containing 0.25 M proline or 0.25 M glycine.
3 shows fragment content measured by SDS PAGE for 8% IgG solution containing 0.25 M proline or 0.25 M glycine.
4 shows the optical density (UV350-500 nm) of two IgG solutions containing 0.25 M proline or 0.25 M glycine.

Example  One

Preparation of Protein Formulations According to the Invention

The starting material in the preparation of intravenous Ig is the approved intermediate of the Kistler Nitschmann ethanol fractionation method. This is the precipitate of the immunoglobulin fraction obtained from plasma using 19% ethanol at pH 5.8.

High molecular weight proteins, lipoprotein complexes and other contaminants were precipitated using octanoic acid and then separated by filtration in the presence of filter aids. The supernatant was then concentrated before being subjected to the low pH incubation step. It was then adjusted to pH 6.5 and the material was further purified by filtration to remove precipitated IgA and IgM. The IgG-rich solution was then finally purified on anion exchange resin according to US Pat. No. 6,093,324 except that the load was 150 g / l (resin).

Virus removal was accomplished using nanofilters.

Formulation : Nanofiltrate was concentrated to 3% protein, diafiltered with 5 volumes of water, and then IgG was concentrated to 120 g / l. Finally, the concentrate was stabilized with 250 mM L-proline, diluted with 100 g IgG per liter and the pH maintained at pH 4.8. The formulated bulk was filtered through a 0.2 μm membrane filter.

Example  2

According to the invention IgG  Test of the formulation

IgG concentrates purified from plasma and inactivated virus were formulated in parallel with precipitation step and chromatography according to Example 1 to 260 ml formulated to pH 4.5, 420 ml formulated to pH 4.8 and pH 5.1. Separated into three fractions of 260 ml. The formulations were then divided, half formulated with 0.25 M glycine and the other half formulated with 0.25 M proline. Final protein concentration was 8% w / v. A 10 ml aliquot was dispensed into 10 ml Type I glass vials (Type I rubber stoppers).

Aliquots were stored at three different temperatures, namely 2-8 ° C, 26 ° C and 45 ° C. Samples at 2-8 ° C. were stored in the presence of light (Phillips TLD 18W / 33). Samples were incubated at 26 ° C. or 45 ° C. for at least 2 months in the dark. The results are shown in Figures 1-4.

Aggregate

Aggregate levels for IgG formulated with glycine were higher under all conditions tested than when formulated with proline.

Significant aggregate formation was promoted by incubation at 45 ° C. This was similar in both proline and glycine formulations. Low pH promoted aggregate formation at 45 ° C. and pH 4.5 formulations containing 12.2% (proline) and 16.7% (glycine) aggregate at 90 days. In contrast, pH 5.1 formulations containing 6.3% (proline) and 8.3% (glycine) aggregate at 90 days.

Dimer

Dimer levels were affected by pH, temperature and excipient type. pH proved to be the most important factor and an increase in dimer levels was observed as the pH of the formulation increased. This was observed in both glycine and proline formulations. These results suggest that formulations containing proline can maintain lower dimer levels compared to glycine formulations. The incubation temperature controls the monomer / dimer equilibrium, with lower temperatures favoring the formation of dimers.

Monomers and Dimer

The monomer / dimer blend content for all formulations at 2-8 ° C and 26 ° C remained above 90%. Due to their higher aggregate content, lower levels were observed in IgG solutions formulated with glycine. Incubation at 45 ° C. resulted in three formulations with levels of 90% or less after 60 days (85.1% glycine, pH 4.5, 89.1 proline, pH 4.5, and 89.1% glycine, pH 4.8). Again, these results highlight the increased proline compared to glycine, the ability to preserve the molecular integrity of IgG molecules.

IgG  snippet

The results suggest that the glycine formulation contains somewhat less levels of fragments than proline. Incubation temperature and pH have proven to be the most important factors affecting IgG fragmentation. At 45 ° C., fragment levels for proline formulations ranged from 5.2% (pH 5.1) to 5.8% (pH 4. 5), while glycine formulations ranged from 4.3% (pH 5.1) to 4.8% (pH 4.8). At elevated pH (4.8 to 5.1), less fragmentation occurred.

Appearance of the solution

Four main parameters were investigated: transparency, turbidity, particles and visible coloring. Parameters such as appearance, transparency and turbidity were satisfactory. Coloring of the solution (yellow / brown) occurred during the incubation period, which was related to both incubation temperature and light exposure. Coloring of the IgG formulation was monitored using an optical density test (UV350-500 nm). Increased pigmentation was associated with light exposure and increased incubation time. Glycine formulations showed 25% to 48% higher optical density than the corresponding proline formulations. These results provide evidence that proline is a better stabilizer than glycine in IgG solutions. At elevated pH (4.8 to 5.1), less pigmentation was achieved than at lower pH (4.5).

Example  3

According to the invention IgG  Stability of the formulation ( pH  Dependencies)

The IgG concentrate purified from plasma and inactivated virus was separated into two fractions in parallel with the precipitation step and chromatography according to Example 1, and 400 mmol / L L-proline at pH 4.2, 4.8, 5.3 and 6.8. Formulated with or without Final protein concentration was 12% w / v. Aliquots of 10 ml were dispensed into glass vials and incubated at 40 ° C. for at least 3 months in the dark. After 0 o'clock and 90 days, the incubated samples were collected by SDS PAGE (fragment) and type B for aggregates, dimer IgG and monomeric IgG by HPLC, for absorbance at 350-500 nm by photometry. Specific antibodies (anti-HB) induced against hepatitis virus surface antigens were analyzed. The results presented in Table 1 show that the best stability of the IgG solution is obtained at weak acidic pH of 4.8 to 5.3.

TABLE 1

Example  4

With different additives Formulated  According to the invention IgG  Formulation Stability

IgG concentrates purified from plasma and inactivated by virus in parallel with the precipitation step and chromatography according to Example 1 were prepared at different pH levels (saccharides and sugar alcohols, amino acids, detergents) at pH 4.2, 4.8, 5.3 and 6.8. Formulated by addition. Final protein concentration was 10% w / v. Aliquots of 10 ml were dispensed into glass vials and incubated at 37 ° C. or 40 ° C. for at least 3 months in the dark. After 90 days, incubated samples were tested for hepatitis B virus surface antigen by ELISA for aggregates, dimer IgG, monomeric IgG and fragments by HPLC, for absorbance at 350-500 nm by photometry. The specific antibodies directed (anti-HB) were analyzed. The results presented in Table 2 show that the best stability of the IgG solution is obtained at weak acidic pH of 4.8 to 5.3 and the most advantageous is the L-proline containing formulation.

TABLE 2a

[Table 2b]

Claims (18)

A stable immunoglobulin formulation having a pH of 4.2 to 5.4, including proline, but no nicotinamide. The formulation of claim 1 comprising proline at a final concentration of at least 0.2M. A stable immunoglobulin formulation having a pH of 4.2 to 5.4, comprising proline at a final concentration of 0.2 M to 0.4 M. The formulation according to any one of claims 1 to 3, wherein the proline is L-proline. The formulation according to any one of claims 1 to 3, wherein the pH is 4.5 to 5.2. The formulation of claim 5 wherein the pH is between 4.6 and 5.0. The formulation of claim 6 comprising proline at a final concentration of 0.25 M. The formulation according to any one of claims 1 to 3, wherein the immunoglobulin concentration is between 5 and 25% w / v. The formulation of claim 8, wherein the immunoglobulin concentration is 15-20% w / v for subcutaneous administration. The formulation of claim 8, wherein the immunoglobulin concentration is 6-15% w / v for intravenous administration. The formulation of claim 10, wherein the immunoglobulin concentration is between 8 and 12% w / v. The formulation according to any one of claims 1 to 3, which is an IgG, IgA or IgM preparation. A pharmaceutical composition comprising an immunoglobulin preparation according to any one of claims 1 to 3 and a pharmaceutically acceptable additive. Providing an aqueous immunoglobulin solution and adjusting the pH of the solution to about 4.2 to 5.4 by adding proline, wherein proline is not used in combination with nicotinamide to stabilize the immunoglobulin formulation. . Reduce the formation of aggregates and / or immunoglobulins in an immunoglobulin formulation, comprising providing an aqueous immunoglobulin solution and adjusting the pH of the solution to about 4.2 to 5.4 by adding one or more stabilizers selected from the group consisting of nonpolar amino acids. Method of reducing the pigmentation of the formulation. The method of claim 15, wherein the nonpolar amino acid is proline. 17. The method of any one of claims 14-16, wherein the pH is adjusted to 4.8. The method according to any one of claims 14 to 16, wherein the proline concentration is adjusted to 0.2 M to 0.4 M.

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